Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Purification of Streptomyces Phospholipase D by Immunoaffinity Chromatoghraphy using Peptide Antibodies

Streptomyces phospholipase D의 정제를 위한 면역친화 크로마토그래피의 개발

  • Park, In-Sun (Division of Biological Sciences, Chonbuk National University) ;
  • Kim, Young-Ah (Division of Biological Sciences, Chonbuk National University) ;
  • Jeong, Su-Jin (Division of Biological Sciences, Chonbuk National University) ;
  • Uhm, Tai-Boong (Division of Biological Sciences, Chonbuk National University)
  • 박인선 (전북대학교 지연과학대학 생물학과) ;
  • 김영아 (전북대학교 지연과학대학 생물학과) ;
  • 정수진 (전북대학교 지연과학대학 생물학과) ;
  • 엄태붕 (전북대학교 지연과학대학 생물학과)
  • Published : 2006.12.30
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Abstract

An immunoaffinity chromatography for the specific binding of Streptomyces somaliensis phospholipase D (PLD) that is considered as an industrially potential enzyme was developed. By using the protein structure prediction programs and the X-ray crystal structure of a Streptomyces PLD, 5 different epitopes with high antigenicity that are predicted to locate on the surface of the S. somaliensis PLD were selected and then synthesized for the preparation of antipeptide antibodies. Each purified rabbit IgG was coupled with NHS-activated Sepharose to prepare the immunoaffinity resins. After one-step purification of the culture concentrate on the antipeptide IgG-coupled Sepharose column, SDS-PAGE and the Western blot analysis of the purified samples showed that purification of PLD on the affinity columns was satisfactory, indicating that the peptide design using the structural information of Streptomyces PLDs was rational. However, the purified PLD in the solution aggregated rapidly, which resulted in poor specific activity and low purification yield.

Streptomyces somaliensis가 생산하는 phospholipase D (PLD)를 정제하기 위하여 펩티드 항체 결합 면역 친화 크로마토그래피용 칼럼을 개발하였다. 단백질 구조 예측 프로그램과 Streptomyces PLD X-선 결정구조를 참조하여, S. somaliensis PLD의 1차 구조로부터 항원특성이 높고. 표면에 위치하는 것으로 예상된 5종류의 펩티드들을 epitope로 선정한 뒤, 이에 대한 항체로 면역친화 크로마토그래피용 칼럼을 제작하였다. 배양 농축액을 칼럼에 통과시켜 정제한 활성 분획을 SDS-PAGE 및 Western blot 결과, 칼럼 종류에 따라 순수한 PLD또는 35 kDa의 단백질 불순물만을 포함하는 PLD 정제 분획을 보여 면역친화 칼럼의 높은 항원결합 특이성을 보여주었다. 그러나 수용액상에서 PLD 자체의 구조적 불안정성 때문에 정제 후 PLD의 특이적 활성 및 정제 수율은 낮았다.

Keywords

References

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